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You searched for +publisher:"Temple University" +contributor:("Walsh, Peter N."). Showing records 1 – 3 of 3 total matches.

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Temple University

1. Shikov, Sergei. Structural Determinants for Heparin Binding in Human Coagulation Factor XI.

Degree: PhD, 2008, Temple University

Biochemistry

Coagulation factor XI plays an important role in the consolidation phase of blood coagulation. Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD~110 nM) affinity via residues (K252, K253 and K255) located in the apple 3 (A3) domain of the molecule. In contrast, the enzyme, factor XIa (FXIa), was shown to bind to heparin with significantly higher affinity (~1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). The interaction between heparin and FXIa potentiates the inhibition of FXIa by protease nexin-2 by 10-fold. In addition, related polyanions heparin and dextran sulfate inhibit the catalytic activity of FXIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI in heparin binding. During this project, wtFXI, FXIR504A, FXIK505A, FXIR507A, FXIR529A, FXIR530A, FXIR532A, and FXIR586A have been expressed and purified. All mutants were homogenous and identical to wtFXI on SDS-PAGE, clotting assays and 1G5 monoclonal antibody binding studied by SPR. In addition, monomeric FXI C321S/K331A was expressed and purified. Utilizing an ELISA assay, no difference in the affinity for heparin between FXIa and FXI was found. Surface plasmon resonance (SPR) data collected for FXI clearly indicate a complex interaction which does not conform to a simple 1:1 Langmuir binding model making it difficult to obtain quantitative information. The complexity of FXI interactions with heparin is likely to arise from the multivalent nature of the binding, in which both protein and heparin have multiple binding sites. Two positively charged residues in the FXI catalytic domain, FXIR507A and FXIR532A, were found to be particularly important for interaction with heparin. The FXIR507A and FXIR532A mutants demonstrated ~ 65% and ~50% decreases respectively in total number of heparin binding sites based on ELISA. Also, the apparent dissociation constants for FXIR507A (KDapp ~13 nM) and FXIR532A (KDapp ~21 nM ) were 6 and 10-fold increased respectively compared with 2.1 nM for the wtFXI. Mutant FXIR586A also demonstrated a defect in affinity (KDapp ~ 13 nM) without an effect on the Bmax. The monomeric FXIC321S/R331A was also characterized for its ability to bind heparin compared with wtFXI. Surprisingly, the monomeric FXI displayed defective binding to heparin according to ELISA (KDapp ~ 30 nM) and SPR methods. Thus, the unique homodimeric structure of FXI in addition to the residues both in its catalytic and A3 domain chains are necessary for high-affinity heparin binding.

Temple University – Theses

Advisors/Committee Members: Walsh, Peter N., Shore, Scott K., Collins, Jimmy H., Suhadolnik, Robert J., Soslau, Gerald.

Subjects/Keywords: Chemistry, Biochemistry; Biophysics, General; Heparin; blood coagulation; ELISA; SPR

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Shikov, S. (2008). Structural Determinants for Heparin Binding in Human Coagulation Factor XI. (Doctoral Dissertation). Temple University. Retrieved from http://digital.library.temple.edu/u?/p245801coll10,13460

Chicago Manual of Style (16th Edition):

Shikov, Sergei. “Structural Determinants for Heparin Binding in Human Coagulation Factor XI.” 2008. Doctoral Dissertation, Temple University. Accessed September 26, 2020. http://digital.library.temple.edu/u?/p245801coll10,13460.

MLA Handbook (7th Edition):

Shikov, Sergei. “Structural Determinants for Heparin Binding in Human Coagulation Factor XI.” 2008. Web. 26 Sep 2020.

Vancouver:

Shikov S. Structural Determinants for Heparin Binding in Human Coagulation Factor XI. [Internet] [Doctoral dissertation]. Temple University; 2008. [cited 2020 Sep 26]. Available from: http://digital.library.temple.edu/u?/p245801coll10,13460.

Council of Science Editors:

Shikov S. Structural Determinants for Heparin Binding in Human Coagulation Factor XI. [Doctoral Dissertation]. Temple University; 2008. Available from: http://digital.library.temple.edu/u?/p245801coll10,13460


Temple University

2. Su, Ya-Chi. LOCALIZATION OF LIGAND-BINDING EXOSITES IN THE CATALYTIC DOMAIN OF FXIa AND DETERMINATION OF THE ROLES OF CALCIUM AND THE HEAVY CHAIN OF FXIa IN FIX ACTIVATION BY FXIa.

Degree: PhD, 2010, Temple University

Biochemistry

Coagulation factor XI (FXI) is a plasma zymogen that is activated to FXIa, the catalytic domain of which contains exosites that interact with its normal macromolecular substrate (FIX), and its major regulatory inhibitor (protease nexin-2 kunitz protease inhibitor, PN2KPI). To localize the catalytic domain residues involved in active site architecture and in various ligand-binding exosites, we aligned the sequence of the FXI catalytic domain with that of the prekallikrein (PK) catalytic domain which is highly homologous (64% identity) in sequence, but functionally very different from FXI. Six distinct regions (R1-R6) of dissimilarity between the two proteins were identified as possible candidates for FXIa-specific ligand binding exosites. FXI/PK chimeric proteins (FXI-R1, FXI-R2, FXI-R3, FXI-R4, FXI-R5, or FXI-R6) containing substitutions with PK residues within the six regions were prepared and characterized. FXIa-R1, R2, R3 displayed enhanced proteolysis after activation by factor XIIa suggesting that the residues within R1, R2 and R3 regions may be important to maintain proper folding of the enzyme. Comparisons of amidolytic assays vs. activated partial thromboplastin time assays showed similar activities for all chimeras except FXI-R6, which displayed 60% of the normal amidolytic activity but only 28% of clotting activity suggesting the possibility that the R6 region (autolysis loop) of FXIa may comprise an exosite involved in the interaction with its macromolecular substrate FIX. This hypothesis was further confirmed by examinations of FIX-activation showing that FIX-activation by FXIa-R6 was significantly impaired compared with that achieved by FXIawt. Although FXI-R5 and FXI-R6 were defective (50-60%) in amidolytic assays, these chimeras were very similar to FXIawt in heparin and high molecular weight kininogen binding assays, suggesting that residues within the R5 and R6 regions are involved in active-site architecture. These chimeras were further investigated to determine whether any of them had acquired kallikrein activity. After activation all except FXIa-R4 showed insignificant activity in assays utilizing a kallikrein-specific chromogenic substrate. FXIa-R4 displayed 87% of the activity of kallikrein using the kallikrein-specific substrate but only 4% of the activity of FXIawt using the FXIa chromogenic substrate. Moreover the cleavage pattern and cleavage rate of high molecular weight kininogen utilizing FXIa-R4 as the enzyme were similar to those achieved with kallikrein but not with FXIawt. Therefore substitutions in the R4 region of FXI with the corresponding residues of PK resulted in loss of activity for the FXIa substrates and gain of activity for the kallikrein substrates suggesting that the R4 region (99-loop) of FXIa plays a role in determining the substrate specificity. The residues of FXIa catalytic domain (R3704, Y5901, E98, Y143, I151, and K192 in chymotrypsin numbering) that are possibly involved in the interactions with its inhibitors have been identified based on the…

Advisors/Committee Members: Walsh, Peter N., Chong, Parkson Lee-Gau, Stitt, Barbara L., Giangiacomo, Kathleen, Sinha, Dipali, Abdel-Meguid, Sherin S..

Subjects/Keywords: Health Sciences; General

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Su, Y. (2010). LOCALIZATION OF LIGAND-BINDING EXOSITES IN THE CATALYTIC DOMAIN OF FXIa AND DETERMINATION OF THE ROLES OF CALCIUM AND THE HEAVY CHAIN OF FXIa IN FIX ACTIVATION BY FXIa. (Doctoral Dissertation). Temple University. Retrieved from http://digital.library.temple.edu/u?/p245801coll10,100396

Chicago Manual of Style (16th Edition):

Su, Ya-Chi. “LOCALIZATION OF LIGAND-BINDING EXOSITES IN THE CATALYTIC DOMAIN OF FXIa AND DETERMINATION OF THE ROLES OF CALCIUM AND THE HEAVY CHAIN OF FXIa IN FIX ACTIVATION BY FXIa.” 2010. Doctoral Dissertation, Temple University. Accessed September 26, 2020. http://digital.library.temple.edu/u?/p245801coll10,100396.

MLA Handbook (7th Edition):

Su, Ya-Chi. “LOCALIZATION OF LIGAND-BINDING EXOSITES IN THE CATALYTIC DOMAIN OF FXIa AND DETERMINATION OF THE ROLES OF CALCIUM AND THE HEAVY CHAIN OF FXIa IN FIX ACTIVATION BY FXIa.” 2010. Web. 26 Sep 2020.

Vancouver:

Su Y. LOCALIZATION OF LIGAND-BINDING EXOSITES IN THE CATALYTIC DOMAIN OF FXIa AND DETERMINATION OF THE ROLES OF CALCIUM AND THE HEAVY CHAIN OF FXIa IN FIX ACTIVATION BY FXIa. [Internet] [Doctoral dissertation]. Temple University; 2010. [cited 2020 Sep 26]. Available from: http://digital.library.temple.edu/u?/p245801coll10,100396.

Council of Science Editors:

Su Y. LOCALIZATION OF LIGAND-BINDING EXOSITES IN THE CATALYTIC DOMAIN OF FXIa AND DETERMINATION OF THE ROLES OF CALCIUM AND THE HEAVY CHAIN OF FXIa IN FIX ACTIVATION BY FXIa. [Doctoral Dissertation]. Temple University; 2010. Available from: http://digital.library.temple.edu/u?/p245801coll10,100396


Temple University

3. Pickens, Brandy S. THE ROLE OF COUP-TFI DURING RETINOIC ACID INDUCED ENDODERMAL DIFFERENTIATION OF P19 CELLS.

Degree: PhD, 2012, Temple University

Biochemistry

ABSTRACT Retinoic acid (RA) is a positive regulator of P19 EC cell differentiation. Pre-B cell leukemia transcription factors (PBXs) act in conjunction with homeobox genes during cell differentiation. PBX mRNA and protein levels are increased rapidly in P19 cells during RA-induced differentiation. However, silencing of PBX expression in P19 cells (AS cells) results in a failure of these cells to differentiate upon RA treatment. Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) and Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) are orphan members of the steroid-thyroid hormone superfamily. The mRNA and protein levels of both COUP-TFI and COUP-TFII are low in proliferating wild type P19 EC cells. However, when wild type P19 cells are induced to differentiate upon RA treatment, COUP-TFI and COUP-TFII mRNA and protein levels are dramatically increased while the levels of pluripotency associated gene products are strikingly reduced. Conversely, COUP-TFI and COUP-TFII mRNA levels fail to be elevated upon RA treatment in PBX AS P19 EC cells. Therefore it was hypothesized that COUP-TFs may be downstream targets of PBX and required factors mediating the RA-dependent differentiation cascade in P19 cells. To determine the role of COUP-TFI during differentiation of P19 cells, PBX AS cells that inducibly express V5 tagged COUP-TFI using the Tet-Off® Advanced Inducible Gene Expression system were prepared. Using this system, we demonstrate that exogenous COUP-TFI expression, in a dose-dependent fashion, leads to growth inhibition, modest cell cycle disruption and early apoptosis. Furthermore, using this cell model which inherently is incapable of undergoing RA-mediated differentiation due to blockage of PBX induction, we demonstrate that a supraphysiological level of COUP-TFI expression can overcome the blockage of RA-dependent differentiation in PBX AS cells. However, AS cells expressing a physiological level of COUP-TFI differentiate to endodermal cells only upon treatment with RA. Additionally, gene expression studies indicate that the reductions of pluripotency maintenance genes observed in the COUP-TFI expressing cells are similar to that of wild type P19 cells (upon RA treatment) suggesting that COUP-TFI expression is a driving force towards loss of pluripotency. Moreover, gene expression studies indicate COUP-TFI is involved in the regulatory modulation of at least two RA response genes, CYP26A1 and HoxA1, indicating that COUP-TFI may have some effect on either maintaining or reducing these genes expression levels when COUP-TFI becomes expressed. COUP-TFII is expressed as two distinct variants, Variant 1(V1) and Variant 2 (V2). V1 is the variant that functions as a classical nuclear receptor by binding target DNA sequences and affecting gene transcription whereas V2 is a truncated form of V1 lacking the ability to bind DNA. We therefore hypothesized that V2 could serve as a dominant negative receptor by limiting the amount of functional V1 in the cell.…

Advisors/Committee Members: Soprano, Dianne R., Soprano, Kenneth J., Walsh, Peter N., Stitt, Barbara L., Masker, Warren, Chong, Parkson Lee-Gau, Gamero, Ana.

Subjects/Keywords: Biochemistry; COUP-TFI; Endodermal Differentiation; P19 Cells; Retinoic Acid

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Pickens, B. S. (2012). THE ROLE OF COUP-TFI DURING RETINOIC ACID INDUCED ENDODERMAL DIFFERENTIATION OF P19 CELLS. (Doctoral Dissertation). Temple University. Retrieved from http://digital.library.temple.edu/u?/p245801coll10,196883

Chicago Manual of Style (16th Edition):

Pickens, Brandy S. “THE ROLE OF COUP-TFI DURING RETINOIC ACID INDUCED ENDODERMAL DIFFERENTIATION OF P19 CELLS.” 2012. Doctoral Dissertation, Temple University. Accessed September 26, 2020. http://digital.library.temple.edu/u?/p245801coll10,196883.

MLA Handbook (7th Edition):

Pickens, Brandy S. “THE ROLE OF COUP-TFI DURING RETINOIC ACID INDUCED ENDODERMAL DIFFERENTIATION OF P19 CELLS.” 2012. Web. 26 Sep 2020.

Vancouver:

Pickens BS. THE ROLE OF COUP-TFI DURING RETINOIC ACID INDUCED ENDODERMAL DIFFERENTIATION OF P19 CELLS. [Internet] [Doctoral dissertation]. Temple University; 2012. [cited 2020 Sep 26]. Available from: http://digital.library.temple.edu/u?/p245801coll10,196883.

Council of Science Editors:

Pickens BS. THE ROLE OF COUP-TFI DURING RETINOIC ACID INDUCED ENDODERMAL DIFFERENTIATION OF P19 CELLS. [Doctoral Dissertation]. Temple University; 2012. Available from: http://digital.library.temple.edu/u?/p245801coll10,196883

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